12/2010 Faculty of Pharmacy Faculty MARKUS HAAPALA University of Helsinki University for Mass Spectrometry Division of Pharmaceutical Chemistry Division Heated Nebulizer Microchips Heated Nebulizer Microchips Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki Helsingiensis in Dissertationes bioscientiarum molecularium Universitatis
MARKUS HAAPALA Heated Nebulizer Microchips for Mass Spectrometry 12/2010 ood-Based Cultures: Expression of Expression ood-Based Cultures: Helsinki 2010 ISSN 1795-7079 ISBN 978-952-10-6257-5 Helsinki 2010 ISSN 1795-7079 ISBN 978-952-10-6257-5 Laccases, Lignin Peroxidases, and Oxalate Decarboxylase Laccases, Lignin Peroxidases, 39/2009 Esko Oksanen Pyrophosphatases - Studies of Soluble Inorganic Enzyme Molecular Choreography 40/2009 Maxim M. Bespalov and Novices GDNF Receptors: Veterans Sten Taina 1/2010 and Androgens Studies on (UGTs); of the Human UDP-glucuronosyltransferases Stereoselectivity Glucuronidation Propranolol 2/2010 Marika Suomalainen Stem Cell Development in Teeth Morphogenesis and of The Signalling Network Controlling Fine-Tuning 3/2010 Kimmo Rantalainen VPg A of Potato virus Biochemical and Structural Properties 4/2010 Sanna Kaivosaari of Drugs and Other Xenobiotics N-Glucuronidation 5/2010 Johanna Mantela of The Inner Ear Role of Cell Cycle Regulators in Development 6/2010 Pinja Jaspers The Through Arabidopsis Thaliana: Regulation and Developmental Responses in Stress RCD1 Factor –Interacting Protein Transcription 7/2010 Marika Pohjanoksa-Mäntylä Special Focus on the Internet and People A and Services for Consumers: Medicines Information Sources with Depression 8/2010 Satu Lakio in Pharmaceutical Powder Technology Understanding of Processes Real-Time Towards 9/2010 Manu Eeva and Their Plant Cell Cultures Angelica archangelica and Plant Secondary Metabolites in Peucedanum palustre 10/2010 Niina Kivikero Atomisation Granulation in Miniaturised Fluid Bed Using Electrostatic Heikkilä Tiina 11/2010 Miniaturization of Drug Solubility and Dissolution Testings Recent Publications in this Series: in Publications Recent Sipilä Timo 31/2009 Ring Aromatic - Sphingomonas for Bioremediation Degradation in Aromatic Plasmids and in Soil and Rhizosphere Cleavage Genes C. Leo 32/2009 Jack Autotransporters on Trimeric and Functional Studies Structural Uutela 33/2009 Päivi and in Studies of Neurotransmitters Spectrometry Mass Liquid Chromatography-Tandem Brain Their Metabolites in the 34/2009 Ranad Shaheen in Food-Borne Illness and Cereulide Spores Bacillus Cereus 35/2009 Katri Berg and Drinking Water in Recreational Associated with Cyanobacteria Bacteria Heterotrophic Aneta Skwarek-Maruszewska 36/2009 Cells Actin Dynamics in Muscle 37/2009 Laura Riihimäki-Lampén Members of the Lipocalin Family with β-Lactoglobulins, Products Interactions of Natural 38/2009 Miia R. Mäkelä W Phlebia radiata and Dichomitus squalens in The White-Rot Fungi Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Finland
Heated Nebulizer Microchips for Mass Spectrometry
by
Markus Haapala
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki, for public examination in Auditorium 1041, Viikki Biocenter 2 (Viikinkaari 5), on May 21st, 2010, at 12 noon.
Helsinki 2010
Supervisors:
Professor Risto Kostiainen Division of Pharmaceutical Chemistry, Faculty of Pharmacy University of Helsinki Finland
Professor Tapio Kotiaho Division of Pharmaceutical Chemistry, Faculty of Pharmacy and Laboratory of Analytical Chemistry, Department of Chemistry, Faculty of Science University of Helsinki Finland
Reviewers:
Professor Janne Jänis Department of Chemistry, Faculty of Science and Forestry University of Eastern Finland Finland
Professor Elisabeth Verpoorte Department of Pharmacy, Faculty of Mathematics and Natural Sciences University of Groningen The Netherlands
Opponent:
Professor Frants Lauritsen Department of Pharmaceutics and Analytical Chemistry Faculty of Pharmaceutical Sciences University of Copenhagen Denmark
© Markus Haapala 2010
ISBN 978-952-10-6257-5 (paperback) ISBN 978-952-10-6258-2 (PDF) ISSN 1795-7079 http://ethesis.helsinki.fi
Helsinki University Print Helsinki 2010
CONTENTS
PREFACE ...... 5
ABSTRACT...... 6
LIST OF ORIGINAL PUBLICATIONS...... 8
ABBREVIATIONS...... 10
1 INTRODUCTION ...... 12
1.1 MINIATURIZATION OF ANALYTICAL DEVICES...... 12 1.1.1 Materials for microfabrication...... 13 1.1.2 Methods for microfabrication...... 14 1.2. ATMOSPHERIC PRESSURE IONIZATION SOURCES AND THEIR MINIATURIZED VERSIONS...... 15 1.2.1 Electrospray ionization ...... 16 1.2.2 Sonic spray ionization ...... 19 1.2.3 Atmospheric pressure chemical ionization...... 19 1.2.4 Atmospheric pressure photoionization ...... 21 1.2.5 Heated nebulizer microchips...... 22 1.3 MINIATURIZATION OF CHROMATOGRAPHY ...... 25 1.4 AMBIENT MASS SPECTROMETRY ...... 26 1.5 TEMPERATURE AND FLUIDIC MEASUREMENTS IN MICROSCALE ...... 27
2 AIMS OF THE STUDY...... 30
3 MATERIALS AND METHODS ...... 31
3.1 CHEMICALS AND MATERIALS ...... 31 3.2 INSTRUMENTATION ...... 33 3.3 MICROCHIP FABRICATION ...... 36 3.3.1 Silicon–glass heated nebulizer microchips...... 36 3.3.2 All-glass heated nebulizer microchips...... 37 3.3.3 Integrated liquid chromatography – heated nebulizer microchips...... 38 3.4 EXPERIMENTAL SETUPS...... 39 3.4.1 Thermal and fluidic measurements...... 39 3.4.2 Chromatography and mass spectrometry...... 41
4 RESULTS AND DISCUSSION ...... 44
4.1 THERMAL AND FLUIDIC PROPERTIES OF HEATED NEBULIZER MICROCHIPS ...... 44 4.1.1 Performance of the thermocouple sensor...... 45 4.1.2 Thermal and fluidic characterization of heated nebulizer microchips...... 45 4.2 DIRECT INFUSION STUDIES ...... 50 4.2.1 Microchip atmospheric pressure photoionization – Fourier transform ion cyclotron resonance mass spectrometry...... 51 4.2.2 Microchip sonic spray ionization ...... 55 4.3 CONNECTING HEATED NEBULIZER MICROCHIPS WITH CHROMATOGRAPHY ...... 56
4.3.1 Gas chromatography – microchip atmospheric pressure photoionization – mass spectrometry ...... 57 4.3.2 Capillary liquid chromatography – microchip atmospheric pressure photoionization – mass spectrometry ...... 59 4.4 INTEGRATION OF CHROMATOGRAPHIC SEPARATION AND HEATED NEBULIZER ...... 61 4.4.1 Microchip structure and operation...... 61 4.4.2. Liquid chromatography ...... 63 4.5 DESORPTION ATMOSPHERIC PRESSURE PHOTOIONIZATION ...... 66
5 CONCLUSIONS ...... 73
REFERENCES...... 75
APPENDIX: ORIGINAL PUBLICATIONS I-VI
PREFACE
This study was carried out at the University of Helsinki, in the Center for Drug Discovery and the Division of Pharmaceutical Chemistry, Faculty of Pharmacy, during the years 2005-2010. Funding was provided by the Graduate School of Chemical Sensors and Microanalytical Systems and the Finnish Funding Agency for Technology and Innovation (projects Biofunctional Microchips nos. 40177/05 and 40380/06 and Microchip based ionization methods for fast mass spectrometric analysis no. 40399/08). I am most grateful to Professors Risto Kostiainen and Tapio Kotiaho for the opportunity to work under their supervision at the Division of Pharmaceutical Chemistry. Whenever there was a problem or an idea to discuss, however large or small, their doors were open. Their enthusiasm for microchip research and mass spectrometry has been an inspiration and their pioneering ideas vital for the results obtained. Warm thanks go to my co-authors for their valuable collaboration and contributions: Ville Saarela and Professor Sami Franssila at the Department of Micro and Nanosciences; Dr Jeremiah Purcell, Dr Ryan Rodgers, Dr Christopher Hendrickson, and Professor Alan Marshall at the National High Magnetic Field Laboratory in Florida; Dr Jaroslav Pól, Dr Tiina Kauppila, Dr Raimo Ketola, and Ville Arvola at the Faculty of Pharmacy; Laura Luosujärvi at the Laboratory of Analytical Chemistry; and Dr Kai Kolari at VTT. Ville deserves special mention: besides doing most of the microchip design work, he spent literally hundreds of hours in the cleanroom fabricating the microchips that were used. He was also a major contributor to the publications, especially the two that we share in our theses. I am indebted to Raimo for his help as a tutor and in the lab. Thanks also to Laura for her contributions, not least the assistance she provided when we began our research. Further, I am indebted to Pekka Östman for introducing me to the world of microchip research. It was great to share in discussions with office mates – Tiina S., Niina, Jarek, Linda, and Mika – about work and many other things. You have been amazing in tolerating my endless questions and always helpful. Thanks go as well to all others in the Division of Pharmaceutical Chemistry – Sirkku, Päivi, and Laura H. in particular – for help and for creating a warm and enjoyable working environment. I am grateful to Professor Janne Jänis (University of Eastern Finland) and Professor Elisabeth Verpoorte (University of Groningen) for their thoughtful reading of the thesis, and to Kathleen Ahonen for improving the English language. Family and friends provided invaluable support. Special thanks are due to Ari and Herkko, two physicists who have triggered amazing and lively discussions about almost anything, especially science, and always dealt kindly with my often odd questions. Warmest thanks go to my parents for providing a supportive and inspiring environment as I grew up. That background has served me well throughout my studies. I am grateful, too, for the help provided by my in-laws. Finally, I express my deepest appreciation to Maria for her support, patience, and love during the past five years and to little Alina for sharing with me the intense joy of life that only a child can feel.
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ABSTRACT
Miniaturized analytical techniques are of increasing interest owing to the faster operation, better performance, ability to analyze much smaller sample volumes, reduced waste production, and lower cost relative to conventional systems. Heated nebulizer (HN) microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically 0.5–10 μL/min, and have previously been utilized for atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Conventional APCI and APPI sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. The requisite for enhancing the performance of HN chips is to understand their principal properties. A new method for thermal and fluidic characterization of miniature gas jets was developed and used to study the heated vapor jet produced by the HN chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. Different types of HN chips were compared and the origins of the differences clarified. The high temperature of the HN microchips allows efficient vaporization of high boiling analytes. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip APPI Fourier transform ion cyclotron resonance mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the ion source and ion inlet of the MS. Microchip APPI provided efficient vaporization of high molecular weight components in crude oil. With the same chip as for APCI and APPI, the first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved simply by applying high (sonic) speed nebulizer gas without heat, corona discharge, or high voltage. SSI significantly broadens the range of applicability of the HN chips, from small stable molecules to labile biomolecules. The performance of the microchip SSI source in terms of flow rate dependence, linearity, limits of detection, and repeatability was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds with capillary LC and other low flow rate separation methods, whereas with electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS with atmospheric pressure ionization showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented GC-μAPPI-MS method for polycyclic aromatic hydrocarbons (PAH) and capillary LC-μAPPI-MS method for steroids showed good quantitative performance. The same high performance microfabrication methods used for the HN microchips were successfully utilized in the fabrication of the first integrated LC–HN microchip. In a single microdevice, there were structures for a packed LC column channel, micropillar frit, channel for optional optical detection, and heated vaporizer. Nonpolar and polar 6
analytes were efficiently ionized by APPI, as demonstrated with PAHs and selective androgen receptor modulators. Ionization of nonpolar and polar analytes had not been possible with previously presented chips for LC–MS since they relied on ESI. With use of APPI-MS, preliminary quantitative performance of the new chip was evaluated in terms of limit of detection, linearity, and repeatability of signal response and retention time. Determination of fluorescent compounds was demonstrated with use of laser induced fluorescence for optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented, and its application to the rapid analysis of compounds of various polarities on surfaces was demonstrated. The DAPPI technique is based on an HN microchip delivering a heated jet of vaporized solvent, e.g., toluene, and a photoionization lamp. The solvent jet is directed toward sample spots on a surface, causing desorption of analytes from the surface. Photons ionize the analytes via reactions similar to those in APPI, and the ions are directed into an MS. The direct analysis of pharmaceuticals from tablets was successfully demonstrated as an application. All in all, the HN microchips were demonstrated to be universal ion sources, which can be used with any API MS, connected to GC or LC, and used with one of several ionization techniques. New integrated LC–HN chip showed good potential for miniaturized LC-MS. The new DAPPI technique was shown to be suitable for rapid analysis of various surfaces and analytes.
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LIST OF ORIGINAL PUBLICATIONS
This doctoral thesis is based on the following six original publications hereafter referred to by their Roman numerals (I-VI):
I Ville Saarela, Markus Haapala, Risto Kostiainen, Tapio Kotiaho, and Sami Franssila. Microfluidic heated gas jet shape analysis by temperature scanning. Journal of Micromechanics and microengineering 2009, 19, 055001.
II Markus Haapala, Jeremiah M. Purcell, Ville Saarela, Sami Franssila, Ryan P. Rodgers, Christopher L. Hendrickson, Tapio Kotiaho, Alan G. Marshall, and Risto Kostiainen. Microchip Atmospheric Pressure Photoionization for Analysis of Petroleum by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Analytical Chemistry 2009, 81, 2799-2803.
III Jaroslav Pól, Tiina J. Kauppila, Markus Haapala, Ville Saarela, Sami Franssila, Raimo A. Ketola, Tapio Kotiaho, and Risto Kostiainen. Microchip Sonic Spray Ionization. Analytical Chemistry 2007, 79, 3519-3523.
IV Markus Haapala, Laura Luosujärvi, Ville Saarela, Tapio Kotiaho, Raimo A. Ketola, Sami Franssila, and Risto Kostiainen. Microchip for Combining Gas Chromatography or Capillary Liquid Chromatography with Atmospheric Pressure Photoionization-Mass Spectrometry. Analytical Chemistry 2007, 79, 4994-4999.
V Markus Haapala, Ville Saarela, Jaroslav Pól, Kai Kolari, Tapio Kotiaho, Sami Franssila, and Risto Kostiainen. Integrated liquid chromatography – heated nebulizer microchip for mass spectrometry. Analytica Chimica Acta 2010, 662, 163-169.
VI Markus Haapala, Jaroslav Pól, Ville Saarela, Ville Arvola, Tapio Kotiaho, Raimo A. Ketola, Sami Franssila, Tiina J. Kauppila, and Risto Kostiainen. Desorption Atmospheric Pressure Photoionization. Analytical Chemistry 2007, 79, 7867-7872.
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Author’s contribution to the publications included in this thesis:
I The experimental work was carried out and the publication written equally by the author and Ville Saarela.
II The experimental work, excluding the microfabrication, was carried out by the author and Dr. Jeremiah Purcell. The article was written by the author with contributions from Dr. Jeremiah Purcell and others.
III The experimental work, excluding the microfabrication, was carried out by the author, Dr. Jaroslav Pól, and Dr. Tiina Kauppila. The article was written by Dr. Jaroslav Pól with contributions from the author and Dr. Tiina Kauppila.
IV The experimental work, excluding the microfabrication, was carried out by the author. The publication was written by the author with contributions from Ville Saarela (microfabrication) and others.
V The experimental work was carried out equally by the author and Ville Saarela. The publication was written by the author and Ville Saarela with contributions from others.
VI The experimental work, excluding the microfabrication, was carried out by the author and Dr. Jaroslav Pól. The publication was written by the author with contributions from others.
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ABBREVIATIONS
APCI atmospheric pressure chemical ionization API atmospheric pressure ionization APPI atmospheric pressure photoionization APTSI atmospheric pressure thermospray ionization ASAP atmospheric solid analysis probe BHF buffered hydrofluoric acid capLC capillary liquid chromatography CE capillary electrophoresis CEC capillary electrochromatography DAPCI desorption atmospheric pressure chemical ionization DAPPI desorption atmospheric pressure photoionization DART direct analysis in real time dc direct current DBE double bond equivalent DESI desorption electrospray ionization DeSSI desorption sonic spray ionization DRIE deep reactive ion etching EASI easy ambient sonic-spray ionization ESI electrospray ionization FEP fluorinated ethylene propylene FT-ICR Fourier transform ion cyclotron resonance GC gas chromatography/gas chromatograph HN heated nebulizer HPLC high performance liquid chromatography IR infrared LC liquid chromatography/liquid chromatograph LIF laser induced fluorescence MALDI matrix assisted laser desorption ionization MEMS microelectromechanical systems MS mass spectrometry/mass spectrometer MS/MS tandem mass spectrometry MSD mass selective detector NC numerical control PA proton affinity PAH polycyclic aromatic hydrocarbon PEEK polyetheretherketone PMMA poly(methyl methacrylate), acrylic glass rf radio frequency RSD relative standard deviation SARM selective androgen receptor modulator SEM scanning electron microscope/microscopy SRM selected reaction monitoring 10
SSI sonic spray ionization TAGA trace atmospheric gas analysis μAPPI microchip atmospheric pressure photoionization μTAS micro total analysis system VUV vacuum ultraviolet
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1 INTRODUCTION
Analytical chemistry studies the composition of materials on the basis of their chemical and physical properties. Besides chemistry itself, analytical methods are important in the biosciences, medicine, environmental research, and many branches of industry. Modern analytical methods commonly involve a separation step, in which the compounds in a sample are separated, and a detection step, in which the separated compounds are detected. One example of this is liquid chromatography-mass spectrometry (LC-MS), in which LC is used for separation and MS for detection. Mass spectrometry is not only a detection method but a powerful analytical technique used widely in many areas of chemistry and other sciences. The first step in measuring the mass spectrum of any sample is ionization. All mass spectrometers work in vacuum, which ranges from 10-3 to 10-10 mbar depending on the type of mass analyzer. Thus the sample has to be either transferred into vacuum before ionization or ionized at atmospheric pressure and the ions then transferred into vacuum. In all mass spectrometers up to the 1980s and in many instruments still today, ionization takes place in vacuum. This creates a limit on the amount of sample that can be transferred from LC to MS because when a liquid vaporizes, its volume expands by a factor of several hundred and a large gas load is created in the vacuum system. Since the LC eluent cannot be pumped directly into the MS, it was for many years impossible to connect LC with MS. The first devices used to couple LC to MS relied on ionization techniques in which ionization took place in vacuum or at significantly reduced pressure. The amount of eluent was reduced, for example, by splitting the liquid flow before ionization to decrease the gas load to the mass spectrometer. The first techniques to become commercial were the moving-belt interface,1 direct liquid introduction,2 thermospray,3,4 and fast atom bombardment.5,6 Of these techniques, thermospray was the most popular and allowed the broad breakthrough of LC-MS as an analytical technique. Today, these techniques are all but obsolete, and commercial LC-MS instruments rely instead on atmospheric pressure ionization methods. All experiments in this work have been performed with use of atmospheric pressure ionization techniques. Vacuum ionization techniques are still popular for other than LC-MS instruments; electron ionization is widely used in gas chromatography–mass spectrometry (GC-MS), for example. Another common vacuum ionization technique, used extensively in the biosciences, is matrix assisted laser desorption ionization (MALDI).
1.1 Miniaturization of analytical devices
Miniaturization of analytical devices has attracted much interest during the last three decades. The general aims of miniaturization are to gain speed; reduce sample, reagent (solvent), and energy consumption; and to enable cost-effective manufacturing of analytical devices. The ultimate goal is a micro total analysis system (μTAS) including, for example, sample preparation, separation, and detection within a single microchip. 12
μTAS is commonly used today to refer to the field of research on miniaturized analytical systems. The materials, or substrates, for microfabrication of microfluidic components and components of microelectromechanical systems (MEMS) are usually in the form of round wafers of 100 or 150 mm size. This is because the tools and methods for microfabrication are largely derived from the semiconductor industry, which also uses wafers. New materials and methods for fabricating specifically microfluidic and MEMS components have also been developed along with the growing interest in the field. The materials and methods for microfabrication relevant in the context of this work are shortly reviewed in the following subsections. A more comprehensive review of microchip-based devices related to mass spectrometry has recently been presented by Sikanen et al.7
1.1.1 Materials for microfabrication
The most common materials for fabrication of microfluidic and MEMS components are silicon, glasses, and polymers. Silicon has excellent properties for MEMS devices including mechanical strength, high thermal conductivity, and adjustable electrical conductivity. In addition, it is compatible with the fabrication of microelectronics, which makes it possible to integrate electronics with mechanical and fluidic components. Thanks to the years of experience with silicon fabrication in microelectronics, a wide variety of fabrication methods are available. Perhaps the most important feature of silicon processing relevant to microfluidics is the ability to fabricate structures with very high aspect ratios (height/width), narrow and deep channels or thin and high micropillars, for example. Some properties may be advantageous or disadvantageous depending on the proposed use. For example, the high thermal conductivity is advantageous if a uniform temperature distribution is required but not if the temperature distribution needs to be localized. The high mechanical strength of silicon allows it to be used as a material for masters in replication methods, but the good electric conductivity makes it impossible to fabricate devices requiring high potential differences, such as electrophoretic separation devices.8,9 Glass encompasses a wide class of materials with essentially limitless composition and properties. However, most glass wafers used in microfabrication are borosilicate glass, quartz, or fused silica. Glass materials are electric insulators and thus good for electrophoretic and other high voltage applications. They are also transparent to visible light, which is ideal for optical detection or visual observation with a microscope. The microfabrication methods for glasses are more limited than those for silicon, and high aspect ratios are difficult to achieve. Glass–glass bonding is easier than silicon–silicon bonding, however, and silicon–glass anodic bonding is a widely applied method for bonding cover wafers to microfluidic and MEMS devices.8 Polymers offer a wide choice of materials and material properties. The fabrication methods for polymers also range widely. In general, polymers are considered to be a simpler and cheaper alternative to traditional silicon and glass. Properties affecting the fabrication and applicability of polymers in microfluidics include glass transition temperature, melting temperature, coefficient of thermal expansion, solvent compatibility, water contact angle, transparency, and porosity.8,10
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1.1.2 Methods for microfabrication
The methods and tools for microfabrication of microfluidic and MEMS come largely from the semiconductor industry, although some processes have been specifically developed. The fabrication is performed in dedicated cleanrooms where temperature, humidity, and especially particle contamination are tightly controlled. In principle, the minimum feature size of the components restricts the fabrication conditions in that possible contaminating particles are ones smaller than the minimum feature size. Thus, many microfluidic devices with feature sizes of roughly 100 μm could be fabricated outside cleanrooms. Nevertheless, this is seldom done since other environmental conditions besides particle contamination are controlled clearly better in cleanrooms. Microstructures are typically created by lithography and etching. In photolithography, a polymeric photoresist is first applied on the substrate and then exposed with UV light through a transparent photomask with opaque patterns. When the resist is developed, a patterned layer of resist is left on the substrate, defining the structures to be created by further processing, typically etching. The photoresist itself can act as a mask for etching or it can be used to pattern another mask, for example, a silicon dioxide thin film hard mask on silicon. Photoresist thickness is usually from hundreds of nanometers to a few micrometers. Some photoresists, such as SU-8, can also be used to create final structures directly by lithography. Advanced lithographic methods can provide pattern sizes well below 100 nm, the costs of equipment and photomasks increasing with decreasing feature size.9 In direct imprinting/replication methods, structures are created by patterning with a 3D shaped mold piece. The patterns on the mold are mirrored to the workpiece (wafer), i.e., an extrusion on the mold makes a channel on the workpiece and vice versa. Imprinting methods include (hot) embossing and casting. In embossing, a mold is pressed with high force against a workpiece, which may be heated. The mold is made of a hard material, typically silicon or metal, and the patterns are transferred onto the softer workpiece. Polymers are common materials amenable to embossing. In casting, liquid monomer is applied on a mold and polymerized, and the mold is detached. A typical material suitable for casting is polydimethylsiloxane (PDMS) elastomer, which thanks to its high elasticity allows even retrograde molds to be used.8,9 Etching methods are classified into wet and dry methods, or into isotropic and anisotropic methods. Wet etching is done in solution and dry etching uses reactive gases in plasma conditions. In isotropic methods etching takes place in every direction at the same rate, while anisotropic methods have directional dependence. Important factors in etching include selectivity and etching rate. Selectivity is the ratio of the etching rate of the mask to that of the material being etched. Some selectivity is always required, and ratios as high as 60,000:1 can be achieved. Etching rate varies widely with the method and material and may be as much as micrometers per minute. Both selectivity and etching rate become critical when high aspect ratios with deep structures are required. Typical examples of isotropic wet etching are glass etching in HF and metal etching with various acids. Anisotropic wet etching can be achieved only with crystalline materials, a common example being silicon etching in KOH. Isotropy in dry etching is controlled by process
14
parameters such as gas composition, flow rate, and pressure, and plasma power. A widely used anisotropic dry etching method is deep reactive ion etching (DRIE) of silicon, which is the method of choice to achieve high aspect ratio silicon structures.8,9 Bonding of wafers is commonly done to achieve closed microstructures. Bonding of dissimilar materials is more challenging than bonding of a material with itself since differences in surface chemistry and thermal expansion need to be taken into account. Bonding may be direct or with an intermediate layer (adhesive) in between. Usually bonding requires that elevated temperature and force are applied. Examples of direct bonding are silicon–silicon fusion bonding at about 1000 °C and glass–glass fusion bonding at about 600 °C (with Pyrex glass). A widely used bonding method with MEMS and microfluidic devices is silicon–glass anodic bonding (field-assisted thermal bonding) at 300–400 °C. A special bonding method is PDMS bonding, which may be either nonpermanent or permanent depending on the surface treatment. In adhesive bonding, an intermediate layer of adhesive material is used to join the wafers. Typical adhesives are photoresist materials or other polymers. The intermediate layer could cause problems in the final device due to differences in surface chemistry and other properties. A nice application is SU-8 adhesive bonding, in which SU-8 wafers are bonded with an SU-8 intermediate layer, resulting in uniform properties of the final structures.8,9
1.2. Atmospheric pressure ionization sources and their miniaturized versions
Atmospheric pressure ionization (API) sources for mass spectrometry were presented in 1958 by Knewstubb and Sudgen.11,12 The first commercial API source for LC-MS was based on work by the group of Horning and Carroll.13-16 A heated vaporizer was used for LC eluent vaporization and corona discharge for ionization. Beta radiation from a 63Ni foil was also used in the early versions. Other API devices for gas analysis17 and LC-MS18,19 and methods for them were subsequently described, while the most successful commercial instrument was the Sciex TAGA (trace atmospheric gas analysis). The real breakthrough in API methods, however, came in the early 1990s as the consequence of developments in electrospray ionization (ESI) and several commercial API instruments presented in the late 1980s. Modern API mass spectrometers are designed so that multiple ionization methods can be applied with the same instrument merely by changing the ion source. To be precise, an ion source is the device that generates the ions, for example an ESI or atmospheric pressure chemical ionization (APCI) source, but in general also the first stages of an API mass spectrometer are considered to be parts of the ion source. Since the pressure difference between the atmosphere and a mass analyzer may be up to 12 orders of magnitude, ions have to be transferred into the mass analyzer by differential pumping through two or more vacuum sections with decreasing pressure. The first transition from atmospheric pressure may be through a small orifice in a plate or a capillary of several centimeters length. The pressure in the first vacuum stage is typically in the order of 0.1– 10 mbar. Various techniques can then be used to transfer the ions further, to the next stage, 15
which may be another pumping stage or the first part of the mass analyzer. An example of an API mass spectrometer including electrospray and differential pumping stages is presented in Figure 1. In this design, a curtain gas is flowing against the spray and the ions to prevent the orifice becoming dirty or clogged. In the following subsections, the most common API methods in use today are shortly reviewed and their principles presented. All API methods can be used for both positive and negative ionization simply by reversing all voltages. With one exception, positive ionization was used in this work and the discussion here is limited to positive ionization.
Figure 1. Schematic view of a typical API mass spectrometer with three pumping stages.
1.2.1 Electrospray ionization
When a high electric potential is applied to a solvent exiting a narrow capillary or a sharp tip, the solvent breaks into thin threads, which fragment further into small droplets. This phenomenon, later called electrospray, was first described by Zeleny in 1914.20 Electrospray ionization as a method for producing large macroions was presented by Dole in 1968.21 At the time, however, API-MS had not been developed. ESI as an ionization method for MS was introduced by Fenn et al.22 in 1984, and Whitehouse et al.23 described an LC-MS interface based on electrospray in 1985. The early ESI interfaces worked only for low μL/min flow rates and thus the pneumatically assisted electrospray (ionspray) of Bruins et al.,24 which allowed flow rates up to 200 μL/min, was an important advance. The final breakthrough of ESI took place after the discovery of its ability to ionize large intact biomolecules (proteins), which made it possible to determine their molecular weight (MW) accurately.25-27 This resulted in a rapid increase in LC-MS applications particularly in the life sciences. The principle of liquid spraying in ESI is illustrated in the upper part of Figure 2. When a high potential is connected to a needle carrying a liquid, the liquid forms a sharp cone, called a Taylor cone, at the end of the needle. In the case of positive potential, the cone is positively charged and the negative charges in the solution migrate to the needle surface. At the tip of the Taylor cone the liquid forms a thin jet, which disintegrates into 16
charged droplets due to Rayleigh instability. These droplets fly away from the needle due to electrostatic forces and undergo processes that finally lead to single gas-phase ions. First, neutral solvent molecules evaporate from the droplets, decreasing the size of the droplets but preserving the charge. This continues until the Rayleigh limit is reached, i.e., the electrostatic repulsion of the charges exceeds the surface tension that holds the droplet together. As a result, the droplet undergoes Rayleigh fission, where it loses a small percentage of its mass but a relatively large percentage of its charge, and several small offspring droplets are produced.
Figure 2. The principle of ESI with ion evaporation and charge residue models presented.
Models called ion evaporation and charge residue have been presented for the final gas-phase ion generation process. The ion evaporation model (Figure 2 top) of Iribarne and Thomson28 assumes that, when a droplet loses solvent by evaporation, the electric field strength on the droplet surface becomes large enough for the droplet to emit ions or solvated ions into gas phase. When an ion is emitted, the charge of the droplet decreases but the mass is practically unchanged. Subsequent evaporation–ion emission cycles follow. The charge residue model (Figure 2 bottom) of Dole et al.,21 on the other hand, assumes that the droplets proceed further through the cycle of solvent evaporation and fission until finally there is just one analyte ion and some solvent in a droplet. The gas- phase ions are formed when the solvent molecules evaporate. It is believed that ion evaporation is the mechanism of ionization for small ions, while the charge residue model applies to large molecules.29,30 The boundary between these mechanisms has been
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suggested to be from a few to several thousand Daltons depending on the type of molecule.31 Practically all commercial ESI sources are designed for joining LC to MS and can handle flow rates up to at least several hundred microliters per minute. As compared with the typical 1–10 μL/min for traditional electrospray, such high flow rates are achieved by assisting the spraying with a high velocity gas flow that promotes nebulization and evaporation of the solvent. The nebulizer can also be heated or an additional heated gas flow may be used to assist the ionization process allowing eluent flow rates as high as 2 mL/min with some instruments. The ionization mechanism of electrospray makes it a concentration-sensitive mechanism. Decreasing the flow rate while keeping the sample amount the same (i.e., increasing the concentration) increases sensitivity and decreasing the flow rate while keeping the sample concentration the same does not affect sensitivity. Electrospray is thus inherently suitable for miniaturization.32,33 In protein characterization, for example, the sample amount may well be very small and a low flow rate ionization method is needed. Electrospray is easily miniaturized by drawing a glass capillary to a very narrow tip, with an i.d. of just a few micrometers. Such narrow tips can be used for analyzing samples of just 1 μL volume, and the flow rate is on the order of tens of nanoliters per minute. The nanospray method, as it is called, was introduced in 1994 by Wilm and Mann.32,33 It is commercially available34,35 and widely used in bioanalysis, especially for proteins. Almost all of the research on miniaturization of API methods for mass spectrometry has concentrated on miniaturization of ESI. This is due to the straightforward fabrication of ESI devices by various microfabrication methods, the advantages of nanospray (reduced flow rate, increased sensitivity), and the similarity of the flow rates of ESI and microfluidic separation methods. In contrast to ESI, APCI and APPI have been developed for connecting traditional LC to MS, and optimal sample flow rates of APCI and APPI are typically in the order of hundreds of microliters per minute. Tens of different microfabricated ESI devices have been presented since the first microchip ESI devices described by Ramsey and Ramsey36 and Xue et al.37 in 1997. ESI chips have been fabricated out of a variety of materials including silicon, glass, and polymers such as PDMS and SU-8. Silicon has been a popular material thanks to its advantageous properties and well-established microfabrication methods. Polymers offer a wide variety of properties and fabrication methods and have also been used extensively. Although glass has good properties, possibilities for microfabrication are somewhat limited and it has been used less than silicon and polymers. The field of microfabricated ESI devices has been reviewed extensively several times. Lazar et al.38 and Koster and Verpoorte39 have reviewed microfluidic devices connected to MS via ESI, with also devices for separation and sample pretreatment integrated with ESI. The microfabrication technologies and microfabricated devices for mass spectrometry, including microfabricated ion sources, were recently reviewed by Sikanen et al.7 from a more general perspective. Microchip-based ESI devices are also available commercially.40,41
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1.2.2 Sonic spray ionization
Sonic spray ionization (SSI), first presented by Hirabayashi et al.42,43 in 1994, is an API method closely related to ESI. During the past ten years, sonic spray has been used in a variety of applications mainly as an alternative ionization method for LC-MS. In SSI, nebulization of solvent is achieved with high pressure and high speed gas flow in a pneumatic nebulizer. The speed of the gas flow in the nebulizer is approximately the speed of sound, hence the name sonic spray. Since neither voltage nor heat is used in SSI, the internal energy of the produced ions is very small, and SSI is an even softer ionization method than ESI.44 In most applications of SSI, some (high) voltage has nevertheless been used, which means that these methods are, strictly speaking, not SSI but closer to ESI. Hirabayashi et al.42 originally suggested that charged droplets are produced in SSI as a result of non-uniform ion concentrations of positive and negative charges in small droplets where the non-uniformity is due to a surface double layer. Later, Takats et al.44 presented results suggesting that charge formation follows Dodd’s statistical charging model45 (which Hirabayashi et al.42 originally ruled out) and that the final ion production takes place through the charge residue model.21 The statistical charging model as it applies to SSI means that the statistical unbalance in natural charge distribution in solution and droplets leads to a net charge in some of the initial droplets. A low flow rate version of SSI, with flow rate down to 100 nL/min, has been reported.46
1.2.3 Atmospheric pressure chemical ionization
Atmospheric pressure chemical ionization for LC-MS was originally reported by Horning et al.14 in 1974, and several applications were demonstrated with their APCI system. Research on APCI was continued at several commercial laboratories, one of the results being the Sciex TAGA system. LC-APCI-MS with a Sciex TAGA was presented by Henion et al.18 in 1982, and high-speed LC-APCI-MS19 was demonstrated in 1986. APCI was widely commercialized in the late 1980s in combination with the API-MS instruments that had been introduced for LC-ESI-MS. Since then, APCI has been widely used for LC- MS, though ESI is still the most popular ionization method. The main working principles of commercial APCI sources are closely similar. The same kind of vaporizers are also used in atmospheric pressure photoionization (APPI) discussed in section 1.2.4 below. Historically, alternative methods for liquid nebulization and vaporization for APCI have been presented, but they are not of interest here. A typical APCI source with heated nebulizer (HN) is presented in Figure 3. The eluent coming into the source is nebulized pneumatically by a high velocity gas flow in a co-axial nebulizer. The eluent capillary is inserted into a larger i.d. capillary, which in some devices is inside a third tubing used for auxiliary gas. After nebulization, the solvent spray is heated causing complete vaporization of droplets. The temperature of the vaporizer is adjustable and may be up to 500 °C. In contrast to ESI, high voltage plays no role in the nebulization process and the produced vapor is neutral. Ionization of the analytes is achieved by means of a corona discharge that initiates gas-phase ionization processes.
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Figure 3. The principle of APCI.
Corona discharge is the electric discharge that may occur in the non-uniform electric field around a high-voltage electrode with small radius of curvature a thin wire or a sharp needle, for example. The counter electrode of the discharge is considered to be large and far away and it plays no role in the discharge process. Corona discharge consists of a corona plasma region close to the curved electrode and a unipolar region further away. The formation of a corona discharge is initiated by an exogenous ionization event (e.g. background radiation) of a neutral atom or molecule in the region of very high electric field strength close to the discharge electrode. A free electron and a positive ion are produced in the ionization. In the case of a positive corona, the discharge electrode is at a positive potential, and the electron is accelerated towards it. The electron undergoes inelastic collisions with neutral species and in the course of these collisions more free electrons and positive ions are produced. The produced electrons undergo the same process, precipitating an electron avalanche towards the electrode. Since all the electrons migrate toward the electrode and ions cannot achieve enough kinetic energy to initiate ionization, the avalanche will collapse after the initial ionization unless more secondary electrons are produced for further avalanches. These are produced in the region outside the corona plasma region by photoionization by photons emitted in de-excitation processes occurring in the plasma and are then accelerated into the plasma region to initiate further avalanches.47 The positive ions produced by corona discharge are the initial source of charge in + + APCI. In air, corona discharge produces mainly N2 and O2 ions. Because of atmospheric moisture and solvent vapor, these ions transfer their charge to water and solvent molecules, and finally protonated solvent molecules or clusters are formed. The most common mechanism for analyte ionization is then proton transfer from protonated solvent to analyte (reaction (1)), which can take place if the proton affinity (PA) of the analyte (M) is larger than that of the solvent (S). This mechanism is highly simplified, it should be noted, since the cluster size of solvent and analyte affects their proton affinities and thereby the whole process. Impurities in the atmosphere and buffers and additives in the LC eluent may also influence the process, complicating it significantly.
+ + (1) SmH + M MH (S)l + Sm-l
The main differences between ESI and APCI are temperature and the ionization process. Ionization in APCI is a chemical gas-phase ion–molecule reaction process, whereas in ESI ionization takes place in the liquid phase. Because ESI is a very soft 20
ionization method and causes minimal fragmentation, even very large labile molecules can be ionized. In APCI, in turn, the high temperature required for vaporization limits its use to relatively stable molecules of MW up to approximately 1000. Although ESI is by far the most widely used ionization technique in API-MS, it nevertheless has some clear limitations. Because the ionization takes place in liquid phase, ESI ionizes effectively only ionic and polar compounds and is ineffective for nonpolar compounds. APCI provides efficient ionization of less polar compounds, in addition to polar and ionic compounds. Moreover, since solvent polarity is a critical factor in ESI, only polar and medium polar solvents can be used. APCI allows the use of nonpolar solvents as well, and can thus be used with normal phase LC, which widens the range of LC applications compared with ESI. Another major issue in ESI is ion suppression due to interfering ions from the sample or eluent, because in the ionization process total ion concentration in the droplets is a key factor. APCI is a gas-phase process and inherently less susceptible to ion suppression.48
1.2.4 Atmospheric pressure photoionization
Atmospheric pressure photoionization was presented in 2000, simultaneously by two independent research groups, as a new ionization method for LC-MS.49,50 The research was motivated by the need to widen the range of compounds analyzable by LC-MS to nonpolar compounds, which are not ionizable with ESI or APCI. Since its introduction, and commercialization in 2000 by Syagen Technologies, APPI has gained interest and has been used in a wide variety of applications. The principles and applications of APPI have lately been reviewed.51,52 In APPI, a co-axial pneumatic nebulizer and heated vaporizer similar to those in APCI are used to vaporize the solvent. Instead of a corona discharge, ionization is achieved by vacuum ultraviolet (VUV) radiation from a krypton discharge lamp. Ionization of analytes (M) takes place by direct photoionization (reaction ((2))) or, more commonly, through gas-phase reactions enhanced with a dopant compound. The latter method is sometimes called dopant-assisted APPI, although the normal term is APPI whether a dopant is used or not.
(2) M + h M + + e-
The original two APPI instruments relied on different VUV lamps, and the difference has continued until now. The PhotoMate APPI source from Syagen uses a radio frequency (rf) excited lamp, which has higher photon output than the direct current (dc) powered lamp used in the PhotoSpray source from Applied Biosystems. In certain applications, the rf excited lamp may enable direct photoionization, but in most cases use of a dopant is advantageous.52 The ionization mechanisms in dopant-assisted APPI have been discussed by Kauppila et al.53 When a dopant (D) is used, the first step in the ionization process is direct photoionization of the dopant (3).
(3) D + h D + + e- 21
After this, there are two common ways to analyte ionization. If the ionization energy (IE) of the analyte is smaller than that of the dopant, charge exchange may take place, producing a radical cation of the analyte (4).
(4) D + + M D + M +
The other path is proton transfer through solvent (S) molecules or clusters. First the dopant donates a proton to the solvent (5), and then the protonated solvent donates a proton to the analyte (6). In this case the PA of the analyte has to be higher than that of the solvent molecule or cluster.